The cellular immune system defense against viruses and tumors depends on the presentation of peptides derived from viral or tumor-specific proteins to T lymphocytes. These peptides must be bound by cell-surface class 1 major histocompatibility complex (MHC) heavy chains to be recognized by lymphocytes. Assembly of the class 1 MHC heavy chains, a light chain called beta 2-microglobulin (beta 2-m) and peptide into the heterotrimeric, complete class I MHC molecule occurs in the endoplasmic reticulum(ER). During class 1 assembly, ER resident proteins such as calreticulin, the transporter associated with antigen processing (TAP), and tapasin interact with peptide-free class 1 heavy chain/beta 2-m. The long-term goals of this study are to understand the mechanism and regulation of the immune response at the level of peptide loading and assembly of the class 1 MHC molecule. The first three studies described in this grant proposal will define the separate roles of each of these ER proteins in the retention of class 1 prior to peptide loading and will probe the nature of their molecular interactions with class 1. Specifically, these studies will 1) determine the importance of class 1 heavy chain glycosylation and the alpha 3 domain for calreticulin association with class 1, 2) define the role of calreticulin in the ER retention of class 1 by the use of cells that over-express, under-express, or do not express calrecticulin, and 3) determine whether TAP, calrecticulin, or tapasin is responsible for the peptide-induced release of class 1 from ER retention. The assembly and surface expression of the class 1 molecule is blocked by several viral proteins. Knowledge of the mechanisms of viral interference in class 1 assembly can reveal a great deal about the normal processes of antigen presentation. One example of such a viral protein is the adenovirus E3-19K protein, which is weakly homologous to members of the immunoglobulin supergene family and so may have an as yet undiscovered cellular homologue. Whether E3-19K displaces peptide or any of the normal ER proteins that chaperone class 1 (calnexin, calreticulin, TAP, or tapasin) is not known. To resolve these issues, the final aim of this proposal is to determine whether the class 1 heavy chains that bind to E3-19K are peptide-occupied and if they have lost association with ER chaperones. In summary, these studies will yield new insights into the regulation of antigen presentation and will be helpful in the future design of rational approaches for clinical treatment of cancer and viral diseases.